What is the difference between microbial identification and profiling? Identification is determining the microbe’s presence or absence in your sample which requires running a No Template Control during your analysis. Profiling is determining the microbe’s relative expression in two or more experimental conditions for which you will need to run a reference sample and a normalizer (provided by QIAGEN).

Search in GeneGlobe

You can search for the following terms:

Entrez Gene IDs (e.g., 835)

RefSeq IDs (e.g., NM_032983, NP_116765)

Gene symbols (e.g., CASP2)

Cat. no. (e.g., SI00299551, QT01342509)

Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)

CpG loci identification numbers (CG#) (e.g., CG17753661)

Do not enter species information in the Search box. Use the drop-down list to select your species of interest.
When searching for miRNAs, do not omit the hyphens. Use hyphens or spaces (e.g., search for hsa-let-7b or hsa let 7b. Do not search for hsalet7b).

This chart demonstrates the difference between the limit of detection (LOD) and the lower limit of quantification (LLOQ). The LOD is defined as the lowest concentration at which 95% of the positive samples are detected, whereas the LLOQ is the lowest concentration that falls within the linear range of a standard curve. LOD depends upon the precision of the assay, and requires at least 40 replicates for determination of a positive sample. For the Microbial DNA qPCR Assays, LLOQ is sufficient to determine assay sensitivity.|This chart shows the distribution of LLOQ for all Microbial DNA qPCR Assays. 93% of all Microbial DNA qPCR Assays have a LLOQ of <100 gene copies. |This chart shows the distribution of LLOQ for microbial identification Microbial DNA qPCR Assays. 92% of all microbial identification assays have a LLOQ of <100 gene copies.|This chart shows the distribution of LLOQ for Microbial DNA qPCR Assays for virulence factor gene detection. 97% of all virulence factor gene assays have a LLOQ of <100 gene copies.|This chart shows the distribution of LLOQ for Microbial DNA qPCR Assays for antibiotic resistance gene detection. 95% of all antibiotic resistance gene assays have a LLOQ of <100 gene copies.|To determine the specificity of Microbial DNA qPCR Assays, each assay was tested against 119 genomic DNA samples from different bacteria and fungi. To facilitate testing, genomic DNA from different microbial species was pooled (10 different genomic DNA samples per pool) and each assay was tested against the different pools. None of the pools contained DNA from the same genus, to facilitate identification of cross-reacting species. Each pool contained the equivalent of 2000 genome copies for each microbial species. In addition, each assay was tested against human, mouse, and rat genomic DNA. A representative example for Streptococcus pyogenes is shown. The assay for Streptococcus pyogenes gave a CT of 26.9 and 26.6 for the Staphylococcus/Streptococcus pool and complete pool [A]. Both pools contained genomic DNA for Streptococcus pyogenes. To determine which genomic DNA was detected by the Streptococcus pyogenes assay, each individual genomic DNA comprising the Staphylococcus/Streptococcus pool was tested separately [B]. Only Streptococcus pyogenes genomic DNA gave an acceptable CT call (26.8) while the others gave a CT>35. Most of the assays were specific as they did not detect unintended targets. For assays that detected other species, the list of detected targets along with in silico predictions are given in the specifications sheet. |To ensure that Microbial DNA qPCR Assays performed comparably in a complex sample, where there may be up to a thousand different microbial species, each assay was tested using stool, tooth plaque, and sputum samples. For each sample, synthetic template targets were spiked in and the CT was compared to synthetic template alone. PCR was performed using several sample types, which included pooled synthetic template targets alone, stool, stool plus pooled synthetic template targets, plaque, plaque plus pooled synthetic template targets, sputum, and sputum plus pooled synthetic template targets. If the CT<35 in stool, plaque, or sputum samples alone, then ΔCT was calculated (i.e., CTstool – CTpooled synthetic template targets). This calculation was performed for all the assays. For each assay, the ΔCT<3, indicating that a complex metagenomic background does not affect the performance of each Microbial DNA qPCR Assay.|

Specificity

Each Microbial DNA qPCR Assay is stringently tested to ensure that it detects only one target species or gene (see figure, Microbial DNA qPCR Assays are highly specific). For assays that do detect more than one target, a list of detected targets and in silico predictions is included on the product sheet.

Microbial DNA qPCR Assays are designed to detect bacterial 16S rRNA gene and fungal ribosomal rRNA gene sequences for species identification, as well as detecting virulence factor genes and antibiotic resistance genes using PCR amplification primers and hydrolysis-probe detection.

Procedure

The procedure for Microbial DNA qPCR Assay Kits is simple, and can be performed in any laboratory with a real-time PCR instrument. DNA is isolated from the sample using the appropriate QIAamp kit, and then PCR reactions are set up using the appropriate Microbial qPCR Mastermix for the PCR instrument. Four separate PCR reactions are prepared for each sample, including Positive PCR Control, No Template Control, and Microbial DNA Positive Control, as well as the Microbial DNA qPCR Assay. Real-time PCR is performed and data is analyzed using web-based data analysis software or Excel templates.

Applications

Microbial DNA qPCR Assay Kits are highly suited for the detection of bacterial or fungal species, or microbial genes for antibiotic resistance or virulence factors, from a variety of samples including stool, sputum, vaginal swab, sewage, and others.